The goal of our study is to develop a new method for purifying and characterizing novel cancer specific DNA with unusual structures that derive from genomic regions with defects in DNA replication control. Accurate duplication of genetic material is essential to maintain genome integrity. To precisely complete the duplication, DNA replication is under strict controls: (1) firing of replication origins only once in a single cell cycle, and () restart/repair replication forks when forks stall or collapse. Defects in replication control could result in either unscheduled replication initiation (re-replication) or incomplete replication with stalled forks. These events are a major cause of genome instability, a major enabling characteristic of tumor proliferation and metastasis. Therefore, an approach that accurately measure and map the region suffering from defects of replication control promotes our understanding of mechanisms underlying genome instability. Both re-replication and replication fork stalling could result in unproductive replication, although little is known about the fate of such unproductive replication. We hypothesize that unproductive replication could result in producing DNAs with unusual structures. Consistent with our hypothesis, we identified such DNAs at a naturally occurring fork stalling site in cancer cells. This finding led us to propose a method that purifies such DNA from cancer cells. By characterizing the DNA using Next Generation Sequencing (NGS), we can systematically determine the genomic locations where the DNAs with unusual structures derive from and thus where DNA replication has problems. Such information is novel and promotes our understanding of defects in replication control in cancer cells. We will develop a method for purifying such DNA by pursuing three aims. Aim 1 will use molecular biology approach to establish a method for enriching DNAs with unusual structures from cancer cells. Aim 2 will use NGS to determine the enrichment of DNA at naturally occurring replication fork stalling sites. Aim 3 will identify DNA with unusual structure derived from replication origins undergoing re-replication. Our experiences in investigating cancer-specific unusual DNA structures in genome-wide support the feasibility of the proposed studies. Successful development of such technology would allow us to investigate the DNA with unusual structures further in large scale studies.